Hammond memorial lecture. New concepts of the control of corpus luteum function

Five new concepts concerning the control of corpus luteum function in the cow have been developed in recent years. Prostacyclin (PGI-2) plays a luteotrophic role. Conversely, products of the lipoxygenase pathway of arachidonic acid metabolism, particularly 5 hydroxyeicosatetraenoic acid (5-HETE), play luteolytic roles. Luteal cells arise from two sources. The small luteal cells are all of theca cell origin; the large cells found early in the cycle (Days 2-6) are mainly of granulosa cell origin. However, a population of large cells found after Day 10 of the cycle are of theca cell origin. Oxytocin of luteal cell origin plays a role in development of the corpus luteum and possibly in its regression. The recently described Ca2+-polyphosphoinositol-protein kinase C second messenger system, as well as the LH-cAMP system, is involved in control of progesterone synthesis in the bovine corpus luteum. Progesterone synthesis in the small theca-derived luteal cells is primarily controlled by the cAMP system. However, elevated intracellular calcium diminishes cAMP-mediated progesterone synthesis in these cells. These findings modify our current concepts of the mechanisms of control of progesterone secretion by the corpus luteum and suggest several new lines of research.     

In-vitro synthesis of a low molecular weight lipid-soluble luteotrophic factor by conceptuses of cows at day 13-18 of pregnancy

Two culture systems for maintenance of Day 13-18 conceptus tissue were developed. Harvested culture media were assayed for luteotrophic activity by determining their ability to stimulate progesterone synthesis by dispersed bovine luteal cells. Significant luteotrophic activity was found in 80% of the 31 tissue culture media studied. A series of experiments carried out to determine the nature of the luteotrophic activity indicate that it is a small (Mr less than 10,000), heat-labile, lipid-soluble substance that is adsorbed by dextran-coated charcoal. The nature and activity of this factor, together with its synthesis by the early bovine conceptus, suggest that it may have a significant role in stimulating progesterone synthesis by the corpus luteum during early pregnancy.     

Morphological characteristics of the bovine corpus luteum during the estrous cycle and pregnancy

The corpus luteum, one of the biological clocks of the estrous cycle and pregnancy, is known foremost for its production of progesterone that blocks the pituitary release of gonadotropins and prepares the uterus for a pregnancy. The cellular sources of this progesterone are the steroidogenic small and large luteal cells. Other luteal cells that are not steroidogenic, but are believed to have an important role in the function of this gland are the fibroblast, macrophages and endothelial cells. The most prominent luteal cell is the large steroidogenic cell characterized by an abundance of smooth endoplasmic reticulum and densely packed spherical mitochondria that are indicative of its contribution to most of the circulating progesterone believed to be constitutively secreted and not under the control of LH. Other distinguishing features of the large luteal cell are the presence of rough endoplasmic reticulum, prominent Golgi, and secretory granules that are indicative of endocrine cells. This cell undergoes dynamic changes across the estrous cycle and pregnancy, believed to reflect a change in progesterone and protein secretion that will eventually influence a successful pregnancy or another ovulation if pregnancy fails. The morphological characteristics of the bovine luteal cells are the focus of this review.     

Thyrotropin stimulates progesterone secretion by luteal cells by activation of the cAMP/protein kinase A signaling system: a potential involvement of protein kinase C

Although the corpus luteum (CL) is not known as a target tissue for thyrotropin (TSH), this hormone increases progesterone production by porcine luteal cells cultured in vitro. In this study we investigated the optimal conditions for TSH-stimulated progesterone secretion as well as the involvement of protein kinase A (PKA) and protein kinase C (PKC) in the mechanism of TSH action on porcine luteal cells. To study the PKA and PKC signaling mechanisms, luteal cells collected from mature CL were incubated with the inhibitor of PKA and potent activators of both kinases: PKA-forskolin and PKC-phorbol ester 12-myriistate-13-acetate (PMA). The PKA inhibitor totally suppressed progesterone production in TSH alone, forskolin alone and in TSH plus forskolin-stimulated luteal cells. Forskolin increased basal (P < 0.05) and TSH-stimulated (P < 0.05) progesterone secretion and cAMP accumulation (P < 0.05). Forskolin and PMA added together to control (non-TSH-treated) luteal cells had an additive effect on progesterone production. In TSH-treated cells, the effect of PMA was statistically significant but did not show an additive effect with forskolin. Further PMA did not affect cAMP accumulation in control and TSH-treated luteal cells. Treatment of control and TSH-treated luteal cells with forskolin and PMA together showed the same increase in cAMP accumulation as with forskolin alone. This is the first demonstration that TSH acts on luteal cell steroidogenesis by activation of the cAMP/PKA second messenger system and also that the PKC signaling pathway may be involved in luteal TSH action on the corpus luteum.     

Arachidonic acid and its metabolites increase cytosolic free calcium in bovine luteal cells

We studied the effects of arachidonic acid and its metabolites on intracellular free calcium concentrations ([Ca2+]i) in highly purified bovine luteal cell preparations. Corpora lutea were collected from Holstein heifers between days 10 and 12 of the estrous cycle. The cells were dispersed and small and large cells were separated by unit gravity sedimentation and flow cytometry. The [Ca2+]i was determined by spectrofluorometry in luteal cells loaded with the fluorescent Ca2+ probe, Fura-2. Arachidonic acid elicited a dose-dependent increase in [Ca2+]i in both small and large luteal cells, having an effect at concentrations as low as 5 microM; and was maximally effective at 50 microM. Several other fatty acids failed to exert a similar response. Addition of nordihydroguaiaretic acid (NDGA) or indomethacin failed to suppress the effects of arachidonic acid. In fact, the presence of both inhibitors resulted in increases of [Ca2+]i, with NDGA exerting a greater stimulation of [Ca2+]i than indomethacin. Prostaglandin F2 alpha (PGF2 alpha) as well as prostaglandin E2 (PGE2) increased [Ca2+]i in the small luteal cells. These results support the idea that arachidonic acid exerts a direct action in mobilizing [Ca2+]i, in the luteal cells. Furthermore, they demonstrate that the cyclooxygenase (PGF2 alpha and PGE2) and lipoxygenase products of arachidonic acid metabolism also play a role in increasing [Ca2+]i in bovine luteal cells. Since the bovine corpus luteum contains large quantities of arachidonic acid, these findings suggest that this compound may regulate calcium-dependent functions of the corpus luteum, including steroid and peptide hormone production and secretion.     

Regulation of the corpus luteum by protein kinase C. I. Phosphorylation activity and steroidogenic action in large and small ovine luteal cells

The activity and steroidogenic action of protein kinase C were evaluated in small and large steroidogenic ovine luteal cells. Protein kinase C activity (per mg protein) was threefold greater in large than in small luteal cells, whereas protein kinase A activity was similar in the two cell types. Phorbol 12-myristate 13-acetate (PMA) activated protein kinase C in luteal cells as demonstrated by membrane association of 91% of available protein kinase C within 15 min of PMA treatment. Longer treatments with PMA produced cells with low protein kinase C activity (protein kinase C-deficient cells) but did not affect cellular viability or protein kinase A activity. Activation of protein kinase C caused an acute, dose-dependent inhibition of progesterone production in unstimulated large and luteinizing hormone (LH)-stimulated small luteal cells. This inhibition by PMA appeared to be specific for protein kinase C since it was greatly attenuated in protein kinase C-deficient cells and since an inactive phorbol ester, 4 alpha-phorbol, had no effect on luteal progesterone production. The inhibitory locus of protein kinase C action in small luteal cells appeared to be distal to the adenylate cyclase enzyme because progesterone production was inhibited similarly in cells stimulated with LH, forskolin, or dibutyryl cyclic adenosine 3',5'-monophosphate. Cholesterol side-chain cleavage activity, as measured by metabolism of 25-hydroxycholesterol, was inhibited by PMA in large, but not in small, luteal cells. These data indicate that activation of protein kinase C specifically inhibits progesterone production in both large and small ovine luteal cells, although the intracellular mechanisms invoked appear to differ in the two cell types.     

Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein is associated with bovine luteal oxytocin exocytosis

The ruminant corpus luteum, in addition to producing progesterone, synthesizes and secretes oxytocin (OT) during the estrous cycle. Secretion of oxytocin occurs by exocytosis of membrane-encapsulated granules of this hormone. Exocytosis of oxytocin involves transport of granules through a cytoskeletal matrix including an actin cortex closely associated with the plasma membrane (PM). Actin filaments crosslinked by various proteins give rise to the structural integrity of the cortex. Myristoylated alanine-rich C kinase substrate (MARCKS), a protein specifically phosphorylated by protein kinase C (PKC), crosslinks actin filaments and anchors the actin network to the inner leaflet of the PM. There is evidence that the intact actin cortex may serve as a barrier, precluding fusion of transport vesicles with the PM. In some secretory cells, phosphorylation of MARCKS has resulted in its translocation from the PM to the cytoplasm with an associated disassembly of the actin cortex. Prostaglandin F(2alpha) (PGF(2alpha)) stimulation of the bovine corpus luteum during the midluteal phase of the estrous cycle activates PKC, which is associated with an increase in OT secretion in vivo and in vitro. Data are presented demonstrating that stimulation of bovine luteal cells with PGF(2alpha) on Day 8 of the cycle promotes rapid phosphorylation of MARCKS protein and causes its translocation from the PM to the cytoplasm and concomitant, enhanced exocytosis of OT. These data are consistent with the premise that MARCKS plays a role in the exocytotic process.     

Functional and morphological characteristics of the first corpus luteum formed after parturition in ewes

The ability of sheep luteal cells from the first corpus luteum formed after parturition (Group F) to secrete progesterone in the presence or absence of LH was compared with that of luteal cells obtained from normal cyclic ewes (Group C). Luteal concentrations of receptors for LH and prostaglandins (PG) F-2 alpha (PGF-2 alpha) and the cellular composition of corpora lutea from Groups F and C were also compared. Luteal cells from Group F secreted less progesterone in either the presence or absence of LH (P less than 0.01). There was no difference in the number of receptors for LH or PGF-2 alpha per luteal cell between Groups F and C (P greater than 0.1), nor was there a difference in the number of large or small steroidogenic luteal cells (P greater than 0.1). It was concluded that, if short-lived corpora lutea are insensitive to gonadotrophins, this response is not mediated by decreased numbers of receptors for LH. In addition, if the first corpus luteum formed post partum in ewes is more sensitive to the luteolytic effects of PGF-2 alpha, this effect is not mediated by an increased number of receptors for PGF-2 alpha or an increased proportion of PGF-2 alpha-sensitive large luteal cells.     

Demonstration of luteinizing hormone/human chorionic gonadotrophin receptor-binding inhibitor in aqueous extracts of frozen human corpus luteum

Aqueous extracts of frozen human corpora lutea were tested for the presence of an inhibitor of luteinizing hormone-receptor site binding (LHRBI) and for the subsequent effect on the stimulatory response of luteinizing hormone (LH) on progesterone synthesis by sheep ovarian cells. In the presence of human corpus luteum extract of normal menstrual cycle (30,000-g supernatant), the binding of 125I human chorionic gonadotrophin (hCG) to granulosa and luteal cells of sheep ovaries was markedly reduced, but the ability of rat testicular LH receptors to bind labelled hCG was less affected. However, extracts of corpora lutea of the first trimester of pregnancy appeared to be less inhibitory on the binding of LH/hCG to ovarian cells and had no effect on the binding of rat testicular cells compared to those of normal menstrual cycle. Addition of both extracts separately inhibited the LH-stimulated in vitro progesterone synthesis by granulosa cell cultures and by incubated sheep corpus luteum slices. These findings provide evidence for the presence of LHRBI in human corpus luteum.     

Purification, morphology, and progesterone production and content of three cell types isolated from the corpus luteum of the sheep

A method is presented for the isolation and purification of three cell types, endothelial cells, small luteal cells and large luteal cells, from the ovine corpus luteum. The method involves enzymatic dispersion of luteal tissue followed by centrifugation of separated cells on a Ficoll gradient. The three purified cell types and others, particularly fibrocytes and smooth muscle cells, that were removed during purification, were identified by their morphology. The cell yield, the cellular composition and cellular progesterone content of each fraction from the Ficoll gradient were measured. The endothelial cell fractions were relatively free of contamination by other cell types and had negligible progesterone. Fractions of small luteal cells and those of large luteal cells contained endothelial cells but were relatively free of other cell types. Large luteal cells contained significantly more progesterone, produced more progesterone when incubated in culture, but were less responsive to luteinizing hormone than small luteal cells.     

Gap junctional intercellular communication of bovine luteal cells from several stages of the estrous cycle: Effects of prostaglandin F2α, protein kinase C and calcium

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The present studies were performed to evaluate the effects of luteotropic and luteolytic hormones, and also intracellular regulators, on contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. Bovine corpora lutea (CL) from the early, mid and late luteal phases of the estrous cycle were dispersed with collagenase and incubated with no treatment, LH, PGF or LH + PGF (Experiment 1), or with no treatment, or agonists or antagonists of protein kinase C (TPA or H-7) or calcium (A23187 or EGTA; Experiment 2). After incubation, media were collected for determination of progesterone concentrations. Then the rate of GJIC was evaluated for small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells by using the fluorescence recovery after photobleaching technique and laser cytometry. Luteal cells from each stage of the estrous cycle exhibited GJIC, but the rate of GJIC was least (P<0.05) for luteal cells from the late luteal phase. LH increased (P<0.05) GJIC between small luteal cells from the mid and late but not the early luteal phase. PGF increased (P<0.05) GjIC between small luteal cells from the mid luteal phase and diminished (P<0.05) LH-stimulatory effects on GjIC between small luteal cells from the late luteal phase. Throughout the estrous cycle, TPA decreased (P<0.05) the rate of GjIC between large and small, and between small luteal cells, and A23187 decreased (P<0.05) the rate of GJIC between large and small luteal cells. LH and LH + PGF, but not PGF alone increased (P<0.05) progesterone secretion by luteal cells from the mid and late luteal phases. Agonists or antagonists of PKC or calcium did not affect progesterone secretion by luteal cells. These data demonstrate that both luteal cell types communicate with small luteal cells, and the rate of communication depends on the stage of luteal development. LH and PGF affect GjIC between small luteal cells during the fully differentiated (mid-luteal) and regressing (late luteal) stages of the estrous cycle. In contrast, at all stages of luteal development, activation of PKC decreases GjIC between small and between large and small luteal cells, whereas calcium ionophore decreases GjIC only between large and small luteal cells. Luteotropic and luteolytic hormones, and intracellular regulators, may be involved in regulation of cellular interactions within bovine CL which likely is an important mechanism for coordination of luteal function.     

Control of Ovarian Function in Domestic Animals

Plasma hormone levels during the estrous cycle of the cow, ewe,and sow have been measured, and the patterns of secretion ofestrogens, progesterone, and luteinizing hormone during thecycle have been related to ovarian changes and other informationconcerning the cycle for each species. Peripheral plasma progesteroneand LH levels are generally inversely related during the cyclein each species, and it seems clear that progesterone exertsa negative feedback on LH secretion in all three species, atleast insofar as the cyclic release of preovulatory amountsof LH is concerned. Peak plasma progesterone levels are highestin the sow, lowest in the ewe, and intermediate in the cow.Plasma LH levels at estrus are highest in the ewe, lowest inthe sow, and intermediate in the cow. Sharp peaks in plasmaLH occur at the onset of estrus in the cow, and a few hoursafter the onset of estrus in the ewe and sow; these peaks areof about 6–8 hr duration. LH exerts a luteotrophic actionon the corpora lutea of all three species, and verylow levelsof LH secretion appear capable of maintaining the corpus luteumin the ewe and cow. There is no good evidence that prolactinis luteotrophic in any of these species. Three peaks of plasmaestrogen levels are seen in the ewe and the cow and these appearrelated to periods of accelerated follicle growth. One peakoccurs early in the cycle and before plasma progesterone levelsrise appreciably and another occurs during the luteal phasejust prior to corpus luteum regression. The third peak occursafter plasma progesterone levels decline and is associated withgrowth of the ovulating follicle. The luteal phase estrogenpeak has not been found in the sow. The rapid rise in bloodestrogens after the corpus luteum regresses facilitates thepreovulatorysurge of LH in all three species. Cyclical regressionof the corpus luteum in all three speciesappears to be underlocal control of the adjacent horn of the uterus. Exogenousestrogens are luteolytic in the cow and ewe, but luteotrophicin the sow. The ovaries of all three species contain very poorlydeveloped interstitial tissue probably because of the neailycomplete dedifftrentiation of the thecal cells during atresia.Thus, these animals lack an important source ofsteroid hormonespresent in the lodents and certain other species.     

Expression of mRNA encoding insulin-like growth factors I and II and the type 1 IGF receptor in the bovine corpus luteum at defined stages of the oestrous cycle

Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.     

A comparison of the effects of cyclooxygenase prostanoids on progesterone production by small and large bovine luteal cells

Highly purified preparations of small and large bovine luteal cells were utilized to examine the effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2) and I2 (PGI2) analog on progesterone production. Corpora lutea were obtained from Holstein heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. Progesterone accumulation was determined in 1 x 10(5) small and 5 x 10(3) large cells after 2 and 4 h incubations respectively. Progesterone synthesis was increased (p less than 0.05) in the small cells by the increasing levels of PGF2 alpha, PGE2, carba-PGI2 and LH. PGF2 alpha, but not PGE2 or carba-PGI2 increased (p less than 0.05) LH-stimulated progesterone production. There was no interaction of various combinations of prostaglandins on progesterone production in the small cells. In the large cells, PGF2 alpha had no effect on basal progesterone production. However, it inhibited LH-stimulated progesterone synthesis. In contrast, PGE2 and carba-PGI2 stimulated (p less than 0.05) basal progesterone production in the large cells. In the presence of LH, high levels of carba-PGI2 inhibited (p less than 0.05) progesterone synthesis. The PGE2 and PGI2-stimulated progesterone production in the large luteal cells was also inhibited in the presence of PGF2 alpha. These data suggest all of the prostaglandins used exert a luteotropic action in the small cells. In the large cells only PGE2 and carba-PGI2 are luteotropic, while PGF2 alpha exerts a luteolytic action. The effects of the prostaglandins in the small and large luteal cells suggest that their receptors are present in both cell types.     

Progesterone production by monkey luteal cell subpopulations at different stages of the menstrual cycle: changes in agonist responsiveness.

Small (less than or equal to 15 microns diameter) and large (greater than 20 microns diam.) luteal cells of the rhesus monkey have been separated by flow cytometry based on light scatter properties. To determine whether the steroidogenic ability and agonist responsiveness of luteal cell subpopulations vary during the life span of the corpus luteum, small and large cells were obtained at early (Days 3-5), mid (Days 7-8), mid-late (Days 11-12), and late (Days 14-15) luteal phase of the cycle. Cells (n = 4 exp./group) were incubated in Ham's F-10 medium + 0.1% BSA for 3 h at 37 degrees C with or without hCG (100 ng/ml), prostaglandin E2 (PGE2; 14 microM), dibutyryl-cAMP (db-cAMP; 5 mM), or pregnenolone (1 microM). Basal progesterone (P) production by large cells was up to 30-fold that by small cells depending on the stage of the cycle. HCG stimulated (p less than 0.05) P secretion by both small (1.8 +/- 0.2-fold) and large (3.7 +/- 0.7-fold) cells in the early luteal phase. HCG responsiveness declined during the luteal lifespan; P production by small cells was not significantly enhanced by hCG by mid luteal phase, whereas that by large cells was stimulated 1.7 +/- 0.2-fold (p less than 0.05) even at late luteal phase. Cell responses to db-cAMP were similar to those for hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

A light and electron microscopic study of the periparturient bovine corpus luteum

Corpora lutea were obtained surgically from fifteen mature Angus crossbred cows representing three experimental groups of five cows each. Cows in Group A were 180 days of gestation, cows in Group B had recently experienced parturition (相似文献   

Differential effects of lysolipids on steroid synthesis in cells expressing endogenous LPA2 receptor

Incubation of ovarian luteal cells with the bioactive lipid mediator lysophosphatidic acid (LPA) for 180 min abolishes gonadotropin-induced steroid production with no attenuation of the cyclic AMP accumulation. Treatment with the lysolipid also diminishes [14C]steroid production in cells preloaded with either [14C]cholesterol or [14C]acetate. Neither the expression of steroidogenic acute regulatory (StAR) protein nor in vitro steroid synthesis is affected in isolated mitochondrial fractions. The LPA-induced attenuation of steroid production occurs only in the mid-cycle corpus luteum and is associated with a transient endogenous expression of mRNA for the lysophosphatidic acid A2 (LPA2) receptor (with no concomitant changes in the expression of LPA1 receptor). Expression of LPA2 is accompanied by LPA-induced sphingosine-1-phosphate (S1P) production. Because luteal cells, in the presence of the sphingosine kinase inhibitor dihydrosphingosine, can overcome the inhibitory effects of LPA on steroid synthesis, we suggest the possible requirement of intracellular S1P production. Interestingly, no LPA-induced inhibition of 8Br-cAMP-stimulated progesterone synthesis can be detected in Leydig tumor cell line MA10 cells expressing only LPA2 receptor. Surprisingly, however, exogenous S1P inhibits agonist-stimulated progesterone in both cell types by inhibiting cyclic AMP accumulation, suggesting different mechanisms of action.